Difference between revisions of "Team:Virginia/Design"

In our mission to create a device to increase metabolic flux, we decided to focus on the resveratrol pathway, one that is simple and researched. To increase pathway flux we propose the use of zinc-finger-domain-containing DNA scaffolds localized to the lumen of (BMCs), to which zinc finger fusion proteins capable of resveratrol biosynthesis are bound .

In order to achieve our goals, we planned to engineer our device to have four main components. These main components include the bacterial microcompartment (BMC), reverse transcriptase (RT), a scaffold DNA template, and scaffold proteins.

The first component is responsible for the production of the bacterial microcompartments. In this design, we propose the use of the Pdu microcompartment which, in nature, encloses enzymes producing 1,2-propanediol in Salmonella enterica. For use in this design only the proteins coding for the shell are required so that the 1,2-propanediol pathway can be replaced by the DNA-scaffolded resveratrol synthesis pathway. For the assembly of an empty Pdu microcompartment shell, it has been shown that only the PduA, PduB, PduJ, PduK, PduN, PduU, and PduT proteins are required forming the complete construct, PduABJKNUT.

The second component is responsible for the production of HIV reverse transcriptase (HIV-RT) and Murine leukemia reverse transcriptase (ML-RT) that aids in the production of DNA scaffolds. While only HIV-RT is needed to produce the scaffolds, it has been seen that the co-expression of ML-RT leads to an increase in DNA production.

The third component is responsible for the RNA templates needed to utilize the enzymes needed for the production of the DNA scaffold as well as its reverse complement. These RNA templates are referred to as r_oligos and consist of the HIV-RT promoter known as the HIV Terminator-Binding Site (HBTS). This HBTS sequence is placed downstream of the scaffold zinc finger domain containing scaffold because HIV-RT processes the template from the 3’ to 5’ direction. There are two r_oligo RNA sequences because each reverse transcriptase produces single stranded DNA so complementary strands need to be produced. Both of these r_oligo RNA sequences are controlled under two copies of the same promoters to control the concentrations of the scaffolds. Lastly, the produced scaffold sequence comprises a domain where the PduD protein will bind and is followed by the 4Cl and STS binding sites.

The fourth component controls the production of the enzymes PduD, 4Cl, and STS that have an added zinc finger domain. All of these proteins are produced by the same part.

These four components will make up the main device we call Manifold. This device will be able to form the BMC-Scaffold complex which will isolate the resveratrol production pathway from the rest of the cell. We delegated design and production of these parts to our wetlab sub teams, categorized the BMC team, DNA team, and Enzyme team. The composite parts of the subteams can be seen in the combined flow chart in the Experiments page.

For our assembly, we mainly will use the Golden Gate Assembly method. We have chosen this assembly for several reasons. The utilizes type IIS restriction enzymes to cut the different parts and form a composite part . The type IIS enzymes are able to cleave outside of their recognition sites and create single-stranded overhangs with non-specific sequences. Since these overhangs are separate from the recognition sequence, they can be customized to direct assembly of multiple DNA fragments. This allows us to determine the direction of the parts and make sure that they are assembled in the correct order. Since the entry and destination vectors have recognition sites that are complementary and in opposite directions, this can create a final plasmid where there is no recognition site. Therefore, once the insert has been ligated, it cannot be cut again. As a result, the ligation process is very efficient and many fragments can be assembled in a single reaction. Additionally, the parts can be designed so that the there are shorter scars or no scars between the parts in the final construct.

To test for the BMCs, we will be using electron microscopy. After transformation with the plasmid, the E. coli will be sectioned and viewed to check for the presence of any BMCs. If BMCs are seen, the bacteria will then be transformed with the previously produced PduD-GFP fusion plasmid and the cells will be viewed under a fluorescence microscope at a resolution of 0.2 μm. In the absence of BMCs, PduD has been shown to localize to the end of E. coli cells, however if they are properly binding to the BMCs then they will be distributed throughout the cytoplasm in a slightly more uniform manner . Additionally, if no fluorescence or continuously uniform fluorescence is seen, then the PduD-GFP fusion steps must be redone. If uniform, discrete patches of fluorescence are seen, we can compare those images to the results of a study. In this study, PduC-GFP, PduD-GFP, and PduV-GFP were individually coproduced with mCherry-labeled BMCs. The image analysis revealed that PduC and PduD were localized within the interior of the microcompartments . By looking at our images and the study’s images, we will be able to see if the red (BMC) and green (PduD or PduA) signals coalesced at the same discrete structures; and that the PduA or PduD are accordingly, localized in the microcompartments as well.

To test that the resveratrol pathway can be implemented with our system, we will be splitting up the assay into three categories. The first assay will comprise of the the 10-beta competent E. coli with only the free enzymes 4Cl-ZF/STS-ZF and the combined 4Cl-ZF/STS-ZF and ACC-ZF/ACS-ZF assembly in order to test for the production of resveratrol as well as acetyl-coA and malonyl-coA which are also intermediates in the pathway. The second assay includes the scaffold and fusion enzymes, the third assay comprises of the entire Manifold system. Afterwards we will be putting these three groups into ethyl acetate to extract resveratrol. Then we will be using to determine the resveratrol yield from the extraction . Finally, we will use to verify the identity of the resveratrol . This assay will help us determine how effectively our parts and Manifold system can affect yield of the resveratrol pathway.